129?A novel CAR conducting antigen-specific JAK-STAT signals demonstrates superior antitumor effects with minimal undesired non-specific activation

摘要

Background Despite recent impressive successes in chimeric antigen receptor (CAR)-T cell therapy, there are still considerable clinical challenges. To improve T cell persistence and antitumor effect, which are critical for clinical responses, various efforts have been made to optimize the CAR design such as the inclusion of a costimulatory domain(s). It is known that non-specific activation of CAR-T cells is greatly influenced by the CAR design, and excessive T cell activation leads exhaustion of T cells and depletion of na?ve/memory subsets important for durable clinical responses. Thus, the CAR construct needs to be optimized so that transduced T cells persist and induce potent antigen-specific response with reduced non-specific activation. For optimal T cell activation and proliferation, three signals including TCR (signal 1), co-stimulatory (signal 2), and cytokine (signal 3) signals, are essential. The conventional second and third generation CARs containing CD3ζ and a co-stimulatory domain such as a signal domain of CD28 and 4-1BB can conduct signal 1 and 2, but not signal 3. Recently, we have developed a new generation JAK-STAT CAR composed of a truncated cytoplasmic domain of the IL-2 receptor β chain and STAT3/5 binding motifs, CD28 co-stimulatory domain, and CD3ζ domain. The novel anti-CD19 JAK-STAT CAR-T cells showed antigen-specific activation of the JAK-STAT signaling pathway, enhanced proliferation, and limited terminal differentiation in vitro compared to second generation 28ζ CAR or 4-1BBζ CAR-transduced T cells. Furthermore, the anti-CD19 JAK-STAT CAR-T cells demonstrated superior in vivo persistence and antitumor effect in mouse models. 1 In addition, we previously showed that a hinge region and the composition of a single chain variable fragment (scFv) such as the order of VH and VL regions critically influence not only antigen-dependent activation but also undesired antigen-independent activation known as tonic signaling. 2 Methods In this study, we have optimized the scFv design in 28ζ CAR and JAK-STAT CAR constructs to show superior antigen-specific activation and reduced tonic signaling for several targets (CD19, CD20, Mesothelin, and GD2). And we have evaluated the feature of JAK-STAT CAR-T cells compared to 28ζ CAR-T cells. Results JAK-STAT CAR-T cells showed superior antigen-specific proliferation with less differentiated status, whereas 28ζ CAR-T cells showed antigen-independent proliferation and displayed higher exhaustion marker expression after repetitive stimulations. Conclusions These results suggest that our JAK-STAT-CARs with enhanced antigen-specific response with minimized tonic signaling targeting various antigens has the potential to demonstrate improved clinical efficacy.